重组蛋白sCAR-TMTP1增强腺病毒对骨肉瘤细胞基因转染效率的研究

目的 提高腺病毒(adenovirus,ADV)在缺乏柯萨奇腺病毒受体(coxsackie and adenovirus receptor,CAR)的骨肉瘤细胞中的转染效率.方法 通过昆虫(Bac-to-Bac)杆状病毒表达系统表达含有CAR胞外段(sCAR)及TMTP1肽的重组蛋白(sCAR-TMTP1),并对其纯化、鉴定.流式细胞仪分别检测骨肉瘤细胞及人胚肾细胞293(HEK293)的CAR表达水平,ADV对其的感染效率及FITC-TMTP1的结合能力.重组蛋白sCAR-TMTP1与ADV共同孵育形成复合物,流式细胞仪研究其在缺乏CAR的骨肉瘤细胞(MG-63)中的转染效率.结果 表达及纯化的sCAR-TMTP1蛋白在相对分子质量约31000处可见特异蛋白条带,感染的sf9细胞裂解上清可与兔抗小鼠sCAR单克隆抗体发生特异性反应.MG-63可与FITC-TMTP1特异结合,其CAR表达水平低,ADV转染效率低.经sCAR-TMTP1修饰后的ADV/GFP对MG-63细胞的感染效率显著高于未经修饰的含报告基因GFP的ADV(ADV/GFP).结论 重组蛋白sCAR-TM-TP1可显著提升ADV对缺乏CAR的骨肉瘤细胞的转染效率.

The recombinant fusion protein sCAR-TMTP1 enhances the gene transfer efficiency of ADV on human osteosarcoma cells

Objective To enhance the transduction efficiency of adenovirus on coxsackievirus and adenovirus receptor (CAR) deficient human osteosarcoma cells. Methods The recombinant protein sCAR-TMTP1 was expressed and purified by Bac-to-Bac system, then identified by SDS-PAGE and Western blotting. The expression level of CAR and the infection efficiency of ADV/GFP in different cell lines (osteosarcoma cells& 293) were detected by flow cytometry (FCM), and the positive rate of FITC-TMTP1 binding to above cell lines was also measured by FCM. MG-63 cells were exposed to Ad/sCAR-ligand complexes mixtures, and the infection efficiency of ADV/GFP was measured by FCM. Results Either of expressed and purified sCAR-TMTP1 showed a specific protein band with a relative molecular mass of 31000, with which the supernatant of lysed sf9 cells infected showed specific reaction with rabbit anti-mouse sCAR monoclonal antibody. FITC-TMTP1 specifically bound to the MG-63 cells with a lower level of CAR and poor infection efficiency of ADV. ADV/GFP modified with sCAR-TMTP1 mediated an obvious enhancements of GFP expression in MG-63, compared with those cells exposed to ADV/GFP. Conclusion The recombinant fusion protein sCAR-TMTP1 has the ability to enhance the gene transfer efficiency of ADV on CAR deficient human osteosarcoma cells.